Tissue-selective targeting of therapeutic agents is an emerging discipline in the pharmaceutical industry. New cancer treatments based on targeting have been designed to increase the specificity and potency of the treatment, while reducing toxicity, thereby enhancing overall efficacy. Mouse monoclonal antibodies (MAb's) to tumor-associated antigens have been employed in an attempt to target toxin, radionucleotide, and chemotherapeutic conjugates to tumors. In addition, differentiation antigens, such as CD 19, CD20, CD22 and CD25, have been exploited as cancer specific targets in treating hematopoietic malignancies. Although extensively studied, this approach has several limitations. One limitation is the difficulty of isolating appropriate monoclonal antibodies that display selective binding. A second limitation is the need for high antibody immunogenicity as a prerequisite for successful antibody isolation. A third limitation is the elicitation in the patient of an immune response against murine antibodies (human anti-mouse antibody-HAMA response) that often results in a shorter serum half-life, and prevents repetitive treatments, thus diminishing the therapeutic value of the antibody. This latter limitation has stimulated interest both in engineering chimeric or humanized monoclonal antibodies of murine origin, and in discovering human antibodies.
There are many factors that influence the therapeutic efficacy of monoclonal antibodies (Mabs) for treating cancer. These factors include specificity of antigen expression on tumor cells, level of expression, antigenic heterogeneity, and accessibility of the tumor mass. Leukemia and lymphoma have been generally more responsive to treatment with antibodies than solid tumors, such as carcinomas. MAbs rapidly bind to leukemia and lymphoma cells in the bloodstream and easily penetrate to malignant cells in lymphatic tissue, thus making lymphoid tumors excellent candidates for MAb-based therapy. An ideal system would entail identifying a MAb that recognizes a marker on the cell surface of stem cells that produce malignant progeny cells.
To aid in the discovery/production of Mabs, phage libraries have been used to select random single chain Fvs (scFvs) that bind to isolated, pre-determined target proteins such as antibodies, hormones and receptors. In addition, the use of antibody display libraries in general, and phage scFv libraries in particular, facilitates an alternative means of discovering unique molecules for targeting specific, yet unrecognized and undetermined, cell surface moieties.
Leukemia, lymphoma, and myeloma are cancers that originate in the bone marrow and lymphatic tissues and are involved in uncontrolled growth of cells. Acute lymphoblastic leukemia (“ALL”) is a heterogeneous disease that is defined by specific clinical and immunological characteristics. Like other forms of ALL, the definitive cause of most cases of B-cell ALL (“B-ALL”) is not known, although in many cases, the disease results from acquired genetic alterations in the DNA of a single cell, causing it to become abnormal and multiply continuously
Acute Myelogenous Leukemia (AML) is a heterogeneous group of neoplasms with a progenitor cell that, under normal conditions, gives rise to terminally differentiated cells of the myeloid series (erythrocytes, granulocytes, monocytes, and platelets). As in other forms of neoplasia, AML is associated with acquired genetic alterations that result in replacement of normally differentiated myeloid cells with relatively undifferentiated blasts, exhibiting one or more type of early myeloid differentiation. AML generally evolves in the bone marrow and, to a lesser degree, in the secondary hematopoietic organs. AML primarily affects adults, peaking in incidence between the ages of 15–40, but it is also known to affect both children and older adults. Nearly all patients with AML require treatment immediately after diagnosis to achieve clinical remission, in which there is no evidence of abnormal levels of circulating undifferentiated blast cells.
To date, a variety of monoclonal antibodies have been developed that induce cytolytic activity against tumor cells. A humanized version of the monoclonal antibody MuMAb4D5, directed to the extracellular domain of P185—growth factor receptor (HER2)—was approved by the FDA and is being used to treat human breast cancer (U.S. Pat. Nos. 5,821,337 and 5,720,954). Following binding, the antibody is capable of inhibiting tumor cell growth that is dependent on the HER2 growth factor receptor. In addition, a chimeric antibody against CD20, which causes rapid depletion of peripheral B cells, including those associated with lymphoma, was recently approved by the FDA (U.S. Pat. No. 5,843,439). The binding of this antibody to target cells results in complement-dependent lysis. This product has recently been approved and is currently being used in the clinic to treat low-grade B-cell non-Hodgkin's lymphoma.
Several other humanized and chimeric antibodies are under development or are in clinical trials. In addition, a humanized Ig that specifically reacts with CD33 antigen, expressed both on normal myeloid cells as well as on most types of myeloid leukemic cells, was conjugated to the anti-cancer drug calicheamicin, CMA-676 (Sievers et al., Blood Supplement, 308, 504a (1997)). This conjugate, known as the drug MYLOTARG®, has recently received FDA approval (Caron et al., Cancer Supplement, 73, 1049–1056 (1994)). In light of its cytolytic activity, an additional anti-CD33 antibody (HumM 195), currently in clinical trials, was conjugated to several cytotoxic agents, including the gelonin toxin (McGraw et al., Cancer Immunol. Immunother, 39, 367–374 (1994)) and radioisotopes 131I (Caron et al., Blood 83, 1760–1768 (1994)), 90Y (Jurcic et al., Blood Supplement, 92, 613a (1998)) and 213Bi (Humm et al., Blood Supplement, 38:231P (1997)).
A chimeric antibody against the leukocyte antigen CD-45 (cHuLym3) is in preclinical phase for treatment of human leukemia and lymphoma (Sun et al., Cancer Immunol. Immunother., 48, 595–602 (2000)) as a conditioning for bone marrow transplantation. In in vitro assays, specific cell lysis was observed in ADCC (antibody dependent cell-mediated cytotoxicity) assays (Henkart, Immunity, 1, 343–346 (1994); Squier and Cohen, Current Opin. Immunol., 6, 447–452 (1994)).
Although these preliminary results seem promising, they have the following limitations. The final product comprises non-human sequences, resulting in a problematic immune response to non-human material, such as HAMA. This HAMA response prevents repetitive treatments and results in a shorter serum half-life for the product. In addition, the above methods allow for the isolation of a single antibody species only, and only allow for the isolation of antibodies against known and purified antigens. Further, these methods are not selective insofar as they allow for the isolation of antibodies against cell surface markers that are present on normal cells as well as on malignant cells.
Thus, a method, which overcomes these above discussed limitations, would be desirable. Further, such method would ideally enable the identification of target ligands or markers on cancer cells or cells involved in mediating metastis of cancer cells, for example. Additionally, such method would also enable the production of antibodies to such targets. Phage display technology appears to offer such abilities.
The use of phage display technology has enabled the isolation of scFvs comprising fully human sequences. For example, fully human antibody against the human TGFb2 receptor based on a scFv clone derived from phage display technology was recently developed. This scFv, converted into a fully human IgG4 that is capable of competing with the binding of TGFb2 (Thompson et al., J. Immunol Methods, 227, 17–29 (1999)), has strong anti-proliferative activity. This technology, known to one skilled in the art, is more specifically described in the following publications: Smith, Science, 228, 1315 (1985); Scott et al, Science, 249, 386–390 (1990); Cwirla et al., PNAS, 87, 6378–6382 (1990); Devlin et al., Science, 249, 404–406 (1990); Griffiths et al., EMBO J., 13(14), 3245–3260 (1994); Bass et al., Proteins, 8, 309–314 (1990); McCafferty et al., Nature, 348, 552–554(1990); Nissim et al., EMBO J., 13, 692 –698 (1994); U.S. Pat. Nos. 5,427,908, 5,432,018, 5,223,409 and 5,403,484, lib.
Using this phage display technology, the inventors of the present invention have identified cell markers present on or cells in diseased or malignant state. Therefore, it is an objective of the present invention to identify peptides and polypeptides that recognize cell markers that are substantially exposed or over-expressed, particularly on or in cells in a diseased or malignant state.
It is a further objective of the present invention to use and expand phage display technology as an aid to identify such peptides and polypeptides.
It is a further objective of the present invention to identify such peptides and polypeptides by immuno-cross-reactivity.
It is a still further objective of the present invention that such peptides and polypeptides be of fully human origin.
It is a still further objective of the present invention that such peptides and polypeptides be isolated against antigens that may not necessarily be immunogenic.
It is a still further objective of the present invention to provide peptides or polypeptides that prevent, retard or cure cancer, particularly blood-related cancers including leukemia or lymphoma.
It is a still further objective of the present invention to provide for local targeting of cancerous cells with such peptides and polypeptides alone, or associated with, or coupled to, an anti-cancer agent and/or a diagnostic label or marker.
It is a still further objective of the present invention to provide a method for producing a targeting agent against desired ligands.
It is a still further objective of the present invention to identify specific motifs that provide for the recognition of cell markers that are over-expressed in the malignant state and that can be used in the construction of a targeting or diagnostic label or marker for an anti-cancer agent.
It is a still further objective of the present invention to provide a composition comprising an effective amount of such peptides, polypeptides or motifs associated with, or coupled to, an anti-cancer agent or to a diagnostic label or marker.
It has been established that scFv penetrate tissues and are cleared from the blood more rapidly than a full size antibody because they are smaller in size. Adams, G. P., et al., Br. J. Cancer 77, 1405–1412 (1988); Hudson, P. J., Curr. Opin. Immunol. 11(5), 548–557 (1999); Wu, A. M., et al., Tumor Targeting 4, 47 (1999). Thus, scFv are often employed in diagnostics involving radioactive labels such as tumor imaging to allow for a more rapid clearance of the radioactive label from the body. A number of cancer targeting scFv multimers have recently undergone pre-clinical evaluation for in vivo stability and efficacy. Adams, G. P., et al., Br. J. Cancer 77, 1405–1412 (1988); Wu, A. M., et al., Tumor Targeting 4, 47 (1999).
Single chain Fv (scFv) fragments are comprised of the variable domains of the heavy (VH) and light (VL) chains of an antibody tethered together by a polypeptide linker. The linker is long enough to allow the (VH) and the (VL) domains to fold into a functional Fv domain enabling the scFv to recognize and bind its target with the similar or increased affinity of the parent antibody. A commonly used linker comprises glycine and serine residues to provide flexibility and protease resistance.
Typically, scFv monomers are designed with the C-terminal end of the VH domain tethered by a polypeptide linker to the N-terminal residue of the VL. Optionally an inverse orientation is employed: the C-terminal end of the VL domain is tethered to the N-terminal residue of VH through a polypeptide linker. Power, B., et al., J. Immun. Meth. 242, 193–204 (2000). The polypeptide linker is typically around twelve amino acids in length. When the linker is reduced to about three to twelve amino acids, the scFvs can not fold into a functional Fv domain and instead associate with a second scFv to form a diabody. Further reducing the length of the linker to less than three amino acids forces the scFv association into trimers or tetramers, depending on the linker length, composition and Fv domain orientations. B. E. Powers, P. J. Hudson, J. Immun. Meth. 242 (2000) 193–194.
Recently, it has been discovered that mulitvalent antibody fragments such as scFv dimers, trimers, and tetramers often provide higher apparent ffinity over the binding of the parent antibody to the target. This higher affinity offers many advantages including ideal pharmaco-kinetics for tumor targeting applications.
The greater binding affinity of these multivalent forms is therefore desirable in diagnostics and therapeutic regimens. For example, a scFv may be employed as a blocking agent to bind a target receptor and thus block the binding of the “natural” ligand. In such instances, it is desirable to have a high affinity association between the scFv and the receptor to decrease chances for disassociation, which may allow an undesirable binding of the natural ligand to the target. In addition, this high affinity is especially critical when the target receptors are involved in adhesion and rolling or when the target receptors are on cells present in areas of high sheer flow, such as platelets.
Therefore, an object of the invention is multivalent forms of Y1 and Y17 scFv. These multivalent forms include, but are not limited to dimers, trimers and tetramers, sometimes referred to herein as diabodies, triabodies, and tetrabodies, respectively.